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The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria Bassiana

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The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria Bassiana ORIGINAL RESEARCH published: 20 July 2021 doi: 10.3389/fcimb.2021.690731 The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria bassiana Elen R. Muniz 1,2,Ca´ rita S. Ribeiro-Silva 2, Walqu´ıria Arruda 3, Nemat O. Keyhani 4 and E´ verton K. K. Fernandes 1,2* 1 Escola de Veterina´ ria e Zootecnia, Universidade Federal de Goia´ s, Goiaˆ nia, Brazil, 2 Instituto de Patologia Tropical e Sau´ de Pu´ blica, Universidade Federal de Goia´ s, Goiaˆ nia, Brazil, 3 Instituto de Cieˆ ncias Biolo´ gicas, Universidade Federal de Goia´ s, Goiaˆ nia, Brazil, 4 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, United States Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress–response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress–response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Edited by: Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout Guilhem Janbon, (DBbmsn2) and complemented mutant (DBbmsn2/Bbmsn2) strains. Reproductive Institut Pasteur, France parameters of R. microplus engorged females treated topically or by an intra-hemocoel Reviewed by: injection of conidial suspensions were assessed. Treated cuticles of engorged females Almudena Ortiz-Urquiza, Swansea University, were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was United Kingdom assessed. Topically treated engorged females showed high mean larval hatching (>84%) Hokyoung Son, D D Seoul National University, in control and Bbmsn2 treatments, whereas treatment with the wild-type or Bbmsn2/ South Korea Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. *Correspondence: Percent control of R. microplus topically treated with DBbmsn2 was lower than in the ´ Everton K. K. Fernandes groups treated with wild-type (56.1%) or DBbmsn2/Bbmsn2 strains. However, no [email protected] differences on reproductive parameters were detected when R. microplus were treated Specialty section: by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. This article was submitted to microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died Fungal Pathogenesis, a section of the journal before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed Frontiers in Cellular and similar conidial germination and formation of pseudo-appressoria on tick cuticle. Infection Microbiology Histological sections of ticks treated with the wild-type or DBbmsn2/Bbmsn2 strains Received: 04 April 2021 Accepted: 18 June 2021 showed fungal penetration through the cuticle, and into the tick interior. Hyphae Published: 20 July 2021 of DBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h Citation: after treatment. Protease activity was lower on tick cuticles treated with DBbmsn2 than Muniz ER, Ribeiro-Silva CS, Arruda W, those treated with the wild-type or DBbmsn2/Bbmsn2 strains. These data show that loss Keyhani NO and Fernandes E´ KK (2021) The Msn2 Transcription Factor of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but Regulates Acaricidal Virulence in the did not interfere with conidial germination, appressoria formation or sporulation on tick Fungal Pathogen Beauveria bassiana. Front. Cell. Infect. Microbiol. 11:690731. cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate doi: 10.3389/fcimb.2021.690731 that the BbMsn2 transcription factor acts mainly during the fungal penetration process Frontiers in Cellular and Infection Microbiology | www.frontiersin.org1 July 2021 | Volume 11 | Article 690731 Muniz et al. BbMsn2 Regulates Virulence to Ticks and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle. Keywords: tick, biological control, entomopathogenic fungi, cuticle, virulence, Beauveria bassiana, Rhipicephalus microplus, Msn2 INTRODUCTION Urquiza and Keyhani, 2015). In entomopathogenic fungi, the transcription factor Msn2 regulates the conidiogenesis of Beauveria bassiana (Hypocreales: Cordycipitaceae) is one of the B. bassiana and Metarhizium robertsii; a knockout strain of each most widely studied entomopathogenic fungi for applied tick species (DBbmsn2 and DMrmsn2, respectively) reduced 43 and 39% control (Kirkland et al., 2004a; Kirkland et al., 2004b; Fernandes the conidial production in comparison to their respective wild-type et al., 2012). The potential of this fungus to control Rhipicephalus strains (Liu et al., 2013). Also, DBbmsn2 and DMrmsn2 strains had a microplus (Acari: Ixodidae) has been shown in laboratory assays, reduced cell tolerance to chemical and environmental stresses. with variable virulence among B. bassiana isolates (Posadas and Decreased virulence of DBbmsn2 and DMrmsn2 against Lecuona, 2009; Campos et al., 2010; Fernandes et al., 2011; Sun Spodoptera litura (Lepidoptera: Noctuidae) second-instar larvae et al., 2013). According to Fernandes et al. (2011), the mean and Tenebrio molitor (Coleoptera: Tenebrionidae) third-instar lethal concentration of B. bassiana to kill 50% (LC50)of larvae was recorded; the LT50 values were 28 and 25% longer R. microplus engorged female can vary from 107 to 109 than the control strains of B. bassiana (5.5 days) and M. robertsii − conidia ml 1. (4.8 days), respectively. In Galleria mellonella (Lepidoptera: fi B. bassiana conidia infect ticks through their cuticle or Pyralidae) larvae, the LT50 signi cantly increased when treated natural openings (Bernardo et al., 2018). The process of fungal with the knockout strain (LT50 =3.4±0.08days)incomparison infection on ticks is thought to be similar to that known for to the wild-type strain (2.71 ± 0.03 days) (Luo et al., 2015). These insects (Arruda et al., 2005), and includes conidial adhesion on data confirmed that Msn2 is critical for entomopathogenic fungal the host cuticle, production of germ tube, differentiation into infection of insects; however, any similar role towards Acari has not appressorium (something not seen for all B. bassiana isolates), been investigated. Furthermore, to date, no histological penetration through the host cuticle by enzymatic action (e.g., investigations related to Msn2 on infection or effects on lipases, proteases and chitinases) and mechanical pressure, and reproduction have been performed on insects or ticks. Overall, growth within the host integument and hemocoel (colonization little is known about the molecular basis of virulence in B. bassiana stage) (Ortiz-Urquiza and Keyhani, 2016). The host dies by towards ticks. Here, we examined the consequences of loss of Msn2 tissue destruction and by action of toxins from fungi (Schrank on B. bassiana infection towards ticks combining genetic and Vainstein, 2010). However, ticks are known to potentially characterization with enzymatic and histological approaches. Our display significantly higher natural resistances to insect data indicate BbMsn2 is critical for penetration but not subsequent pathogenic fungi, and acaricidal specificfactorsmaybe growth and proliferation once the tick cuticle has been breached. produced by these fungi (Kirkland et al., 2004a, Kirkland et al., Furthermore, important effects were seen with respect to lowered 2004b, Kirkland et al., 2005). female fertility, indicating potential added benefits in the application As mentioned, the virulence of B. bassiana is influenced by of B. bassiana for tick control. the production of proteases, particularly the subtilisin-like protease called Pr1 (Joshi et al., 1995), chitinases and lipases (Fang et al., 2005) and, specifically against ticks, the metabolite oxalic acid has been shown to be important (Kirkland et al., MATERIAL AND METHODS 2005). In the last fifteen years, many studies have contributed to understanding the network of genes which are related to the Beauveria bassiana Strains, Conidial virulence of B. bassiana (Jin et al., 2010; Ortiz-Urquiza and Suspensions, and Viability Keyhani, 2013; Wang et al., 2013; Valero-Jiménez et al., 2016). The strains of B. bassiana knockout DBbmsn2 and complemented Fang et al. (2005) demonstrated that overproduction of the (DBbmsn2/Bbmsn2) were constructed and initially characterized Bbchit1 chitinase enhanced the virulence of B. bassiana against by Luo et al. (2015). DBbmsn2 and the complemented strain were fi aphids, as indicated by the signi cantly lower LC50 and mean obtained from B. bassiana Bb0062. Beauveria bassiana strains lethal time to kill 50% (LT50) of target insects of the mutant strain were cultivated on potato dextrose agar (PDA, Difco Laboratories, − compared to the wild-type strain. Another study using hybrid Sparks, MD, USA) supplemented with 1 g L 1 yeast extract ™ chitinase gene (Bbchit1–BmChBD) showed a 23% reduction of (Bacto Yeast Extract, Sparks, MD, USA) (PDAY) in Petri the LT50 to kill aphids (Myzus persicae) when they were treated plates (90 × 15 mm) and incubated in the dark for 15 days at with the mutant strain of B. bassiana in comparison to its wild- 26 ± 1°C and relative humidity (RH) ≥90%. Temperature and type (Fan et al., 2007). relative humidity in the incubator were monitored with a data ® Transcriptional
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